Recombinant Human TSH Fails to Induce the Proliferation and Migration of Papillary Thyroid Carcinoma Cell Lines

Simple Summary Serum TSH suppression in the management of PTC patients will inevitably cause iatrogenic thyrotoxicosis. Our study challenges, for the first time, the role of human recombinant thyrotropin (rh-TSH) as a mitogen on PTC cell lines. For this study, both K1 and TPC-1 cells were treated with clinically relevant rh-TSH under various conditions. The expression levels of TSHR and thyroglobulin (Tg) were determined and, subsequently, the proliferation and migration were assessed. Additionally, cells were engineered to overexpress TSHR in order to multiply TSH signal transduction. Under the conditions employed, rh-TSH was inadequate for inducing either the proliferation or the migration rate of both transformed and non-transformed cells, while Tg expression was increased. We report that, clinically speaking, rh-TSH doses cannot induce proliferation and migration in PTC cell lines, thus questioning TSH suppression in PTC patients. Further research is warranted to dissect the underlying mechanisms. These results could translate into better PTC patient management. Abstract Thyrotropin (TSH) suppression is required in the management of patients with papillary thyroid carcinoma (PTC) to improve their outcomes, inevitably causing iatrogenic thyrotoxicosis. Nevertheless, the evidence supporting this practice remains limited and weak, and in vitro studies examining the mitogenic effects of TSH in cancerous cells used supraphysiological doses of bovine TSH, which produced conflicting results. Our study explores, for the first time, the impact of human recombinant thyrotropin (rh-TSH) on human PTC cell lines (K1 and TPC-1) that were transformed to overexpress the thyrotropin receptor (TSHR). The cells were treated with escalating doses of rh-TSH under various conditions, such as the presence or absence of insulin. The expression levels of TSHR and thyroglobulin (Tg) were determined, and subsequently, the proliferation and migration of both transformed and non-transformed cells were assessed. Under the conditions employed, rh-TSH was not adequate to induce either the proliferation or the migration rate of the cells, while Tg expression was increased. Our experiments indicate that clinically relevant concentrations of rh-TSH cannot induce proliferation and migration in PTC cell lines, even after the overexpression of TSHR. Further research is warranted to dissect the underlying molecular mechanisms, and these results could translate into better management of treatment for PTC patients.


Introduction
Thyroid cancer is the most common endocrine malignancy, accounting for 88% of all endocrine carcinomas and 3% of all human cancers [1][2][3].Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer, accounting for 84% of all cases [4].In the management of PTC patients, a degree of serum thyroid-stimulating hormone (TSH) suppression is required, depending on disease severity and their response to therapy [1,5].Thus, a substantial number of PTC patients are subjected to iatrogenic subclinical hyperthyroidism, which is known to have adverse effects on cardiac structure and function, as well as bone health [6][7][8][9].The beneficial effects of suppressed TSH on the medical outcomes of PTC patients are not well-established [10].In one prospectively followed multi-institutional registry study from the National Thyroid Cancer Treatment Cooperative Study Group in North America on 2936 differentiated thyroid carcinoma (DTC) patients of all stages, it was found that TSH suppression improves overall survival (OS) and disease-specific survival (DSS), but only in high-risk patients [11].Additionally, maintaining a subnormal to undetectable TSH range in stage II patients has a beneficial effect on OS [11].Conversely, in one randomized controlled trial that included 441 patients with DTC at all stages, disease-free survival (DFS) was not more than 10% less than that for patients without TSH suppression [12].In a very recently published systematic review and meta-analysis, the beneficial effects of suppressed serum TSH in intermediate-and high-risk patients were questioned.Nine studies were selected, and in seven of them, progression-free survival (PFS), DFS, and relapse-free survival (RLFS) were similar among patients with suppressed or not-suppressed TSH.In four of the studies, the OS and DSS composite outcomes did not differ between the two groups of patients [13].It is worth mentioning that there is heterogeneity among studies regarding the definition of TSH suppression, ranging from <0.01 to <0.5 µU/mL or lower than the reference range, whereas 'not suppressed' indicates up to 2 µU/mL.The rationale for TSH suppression in the management of PTC patients comes from the notion that TSH, via its receptor, is expressed in the follicular epithelial cells of the thyroid gland and regulates thyroid function and growth [14].The thyroid-stimulating hormone receptor (TSHR) couples to the G proteins of all four subfamilies: these include the stimulatory G protein Gs, which activates adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP); Gi, which inhibits cAMP production; G13, which activates p44/42 mitogen-activated protein kinases (MAPKs-commonly known as ERK1/2); and Gq/G11, which activates phospholipase C to produce inositol-1,4,5-trisphosphate, which is rapidly degraded to inositol monophosphate (IP-1; phosphoinositide signaling).From another perspective, the activating mutations of either TSHR or Ga proteins that underlie functioning thyroid nodules preclude thyroid cancer [1,15].Similarly, autoantibodies against TSHR in Graves' disease are not widely recognized as a risk factor for thyroid cancer development [15,16].Nevertheless, intriguing findings from a mouse model carrying a patient-derived constitutively active TSHR D633H mutation reveal a different scenario [17]; large papillary thyroid tumors emerged in mice carrying the mutation at around one year of age, with nearly all homozygous mice affected.Remarkably, the most commonly observed driver mutations of BRAF, NRAS, and KRAS were notably absent in these tumors.Thus, it seems that enhanced TSHR signaling due to some rare TSHR-activating mutations could lead to thyroid cancer development and growth.However, in the most common thyroid cancers due to BRAF, NRAS, and KRAS mutations, the role of TSHR signaling in the development and/or growth of these tumors has not been extensively studied.Hence, the role of TSH suppression in all these cases warrants investigation.
A few in vitro studies of various thyroid cell culture systems treated with different doses of cattle/bovine TSH (b-TSH) give inconclusive results regarding the proliferation and function of thyroid cells [18][19][20][21][22][23].The doses of TSH used in these studies were extremely high compared to the circulating concentrations found in humans.Recently, recombinant human thyrotropin (rh-TSH) became available on the market.Human TSH has 88% homology with its bovine counterpart (NCBI Homologene).Bovine TSH contains positively charged amino acid residues at the α-subunit, which are known to increase hormone-binding properties compared to human TSH [24].The biological and immunological activities of rh-TSH were examined in human fetal thyroid cells [25].Its biopotency was estimated to be 4.3 ± 0.7 IU/mg, with the immunopotency at 10.2 ± 0.7 IU/mg, whereas the immunopotency of b-TSH is 20-40 IU/mg and is highly dependent on purification [26].
Herein, we investigated for the first time the effects of rh-TSH in physiologically relevant doses that resemble the TSH concentrations in human plasma on the function and proliferation of PTC human thyroid cell lines.For this purpose, we selected two well-established cell lines, K1 and TPC-1, both of which effectively model PTC cells.These cell lines retain their differentiation by expressing TSHR and Tg, classifying them as differentiated PTC cells and indicating their differentiation status [27,28].TPC-1 cells represent a well-differentiated model of PTC and harbor RET/PTC1 fusion [29].RET/PTC alterations are found in 10-30% of PTC patients [30].K1 cells also model well-differentiated PTC, are heterozygous for the BRAF V600E mutation, and possess the R213R mutated p53 (TP53) gene [29].The BRAF V600E activation mutation is the most prevalent molecular alteration in PTC, accounting for 18-87% of all cases [31].
TSHR is known to be a key protein in the signal transduction pathways that control TSH-dependent cell proliferation, migration, and differentiation.Hence, to emphasize and fully assess the role of TSHR activation by rh-TSH in PTC cell proliferation, migration, and Tg synthesis, we created cells that controllably overexpressed the receptor.TSH signaling is also known to be dependent on IGF1R activation since both receptors exhibit significant synergy regarding downstream signal transduction [32].Therefore, to fully assess the effects of TSHR stimulation, not only did we transform the cells to overexpress the receptor but we also co-administered rh-TSH with recombinant human insulin (rh-INS), which can cause IGF1R stimulation [33][34][35].Nonetheless, in most of the in vitro settings reported, TSHR expression is significantly downregulated, a fact that possibly leads to misleading information regarding its actual role.Therefore, to multiply the incoming mitogenic signals, we used an inducible gene expression system to overexpress the receptor and assess its role under conditions of both baseline expression and overexpression.

Thyroglobulin (Tg) and TSHR Expression Determination Using a Quantitative Polymerase Chain Reaction (qRT-PCR)
To assess the baseline expression levels of thyroglobulin (Tg) and TSHR, as well as Tg induction as a result of rh-TSH stimulation in K1 and TPC-1 cells, qRT-PCR was used.Cells were treated with designated rh-TSH doses, then, 24 h later, total RNA was extracted using the Monarch ® Total RNA Miniprep Kit (New England Biolabs, Ipswich, MA, USA).The extracted RNA was eluted in 50 µL of nuclease-free water and treated with Qiagen DNAse I (Germantown, MD, USA), following the manufacturer's procedure.The RNA quantity was assessed using the NanoDrop Q500 spectrophotometer (Quawell, CA, USA).RNA quality was assessed by determination of the ratio for absorbance at 260 nm vs. absorbance at 280 nm (A260 nm/A280 nm), using the NanoDrop Q500 spectrophotometer (which was assessed by calculating the ratios of absorbance for A260 nm/A280 nm and A260 nm/A230 nm).RNA integrity was roughly verified by electrophoresis on a 1.2% denaturing agarose gel stained with ethidium bromide.Then, 1 µg of total RNA was reversetranscribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Life Sciences, Penzberg, Germany), according to the manufacturer's instructions.The cDNA samples were subjected to quantitative PCR (qRT-PCR), which was performed with the LightCycler ® 2.0 Real-Time PCR System (Roche Life Sciences, Penzberg, Germany) using LightCycler ® FastStart DNA Master SYBR ® Green I (Roche Life Sciences, Penzberg, Germany).The cycling conditions consisted of 15 min of denaturation at 95 • C, followed by 45 cycles of 95 • C for 10 s at 60 • C for 30 s, then at 72 • C for 20 s.
The primers used in qRT-PCR for Tg mRNA detection were: The primers used for TSHR mRNA expression were: TSHR forward 5 ′ -GGAATGGGGTGTTCGTCTCC-3 ′ TSHR reverse 5 ′ -GCGTTGAATATCCTTGCAGGT-3 ′ As a reference gene, ACTB (β-actin) was used, which was detected with the following primers: β-actin forward 5 ′ -GCACAGAGCCTCGCCTT-3 ′ β-actin reverse 5 ′ -GTTGTCGACGACGAGCG-3 ′ All primers were purchased from IDT (Leuven, Belgium).The DNA polymerase FIREPol ® and the master mix were from Solis BioDyne (Tartu, Estonia).The specificity of the PCR product was verified by melting curve analysis (single-peak).For qRT-PCR analysis, the data were analyzed using the relative quantification method.The efficiency of each PCR reaction was calculated by serial dilutions of pooled cDNA samples [36].

Treatment with Recombinant Human TSH and/or Recombinant Human Insulin
Equal numbers of K1 or TPC-1 cells (25,000) were seeded on 48-well TC-treated plates (Greiner Bio-One, Kremsmünster, Austria) and left for 24 h to settle.Then, the cells were rinsed twice with a PBS solution and treated with recombinant human TSH (rh-TSH), in the form of Thyrogen ® (Sanofi Genzyme, Cambridge, MA, USA), and/or recombinant human insulin (rh-INS), in the form of Humalog ® (Eli Lilly and Company, Indianapolis, IN, USA).Increasing concentrations were used (as indicated in the relevant figures) in the presence of 2.5% FBS for 48 or 72 h (5-1000 µIU/mL).The FBS concentration of 2.5% was chosen based on preliminary experiments as the minimum (from a range of 1-10%) that would allow the cells to survive and replicate as expected.Simultaneously, using a minimal FBS concentration was crucial to avoid stimulation of the cells by the significant amounts of hormones contained in the bovine serum (endogenous b-TSH, bovine insulinlike growth factor 1 (b-IGF1), insulin (b-INS), and relative co-factors) since the purpose was to administer controlled doses of TSH and insulin.A negative control (vehicle-treated cells) was included in each experiment (culture medium supplemented with 2.5% FBS, without additional rh-TSH/rh-INS), as well as a positive control with an elevated FBS concentration of 10%.FBS could not have been omitted due to the thyroid cells' need for growth factors and iodine salts [37].To maintain the thyroidal characteristics of the cells (avoiding de-differentiation), allow them to proliferate (as all the aforementioned factors are needed), and suppress the apoptosis signals caused by the lack of proper hormonal stimulation, this minimal FBS concentration was necessary and was, thus, maintained during all assays.A detailed description of the FBS concentration selection is presented in Appendix A.

Crystal Violet Assay
Following treatment with the hormones (rh-TSH, rh-INS) for 48-72 h, the final number of viable cells was calculated using the crystal violet assay [38].The culture media were aspirated, the cells were rinsed twice with a phosphate-buffered saline (PBS) (pH = 7.4) solution and were then fixed for 10 min at 25 • C in a 3.7% formaldehyde solution in PBS.Then, the fixation was aspirated, the cells were rinsed, and were subsequently stained in a methanolic crystal violet solution (Sigma-Aldrich, Burlington, MA, USA) for 20 min at 25 • C. Following staining, the cells were rinsed three times with distilled water to remove any residual stain.Then, the water was carefully aspirated, and a 30% acetic acid solution was added to extract the cell-bound crystal violet dye by mild shaking for 20 min at 25 • C. Finally, the extracted solution was transferred to a 96-well flat-bottomed plate (Greiner Bio-One, Kremsmünster, Austria) for spectrophotometry at 590 nm using a microplate reader.The equation used to convert the optical density (OD) to a number of cells (N) was the following: n = 0.5 × 10 5 × OD.This equation was based on preliminary experiments with an increasing number of cells stained using the above-mentioned method.

Wound Healing Assays of K1 and TPC-1 Cells
The wound-healing assay was used as a rapid way to assess the hormones' effects on the migratory potential of both K1 and TPC-1 cells and has also been employed by other researchers [39,40].Cells were seeded in a 6-well plate (Greiner Bio-One, Kremsmünster, Austria) until the formation of a complete monolayer.A scratch was then formed in the cells' monolayer, then, after washing away the scraped cells with a PBS solution, the cells were incubated with rh-TSH concentrations of 5, 10, 20, 50, and 100 µIU/mL and 2.5% FBS for 72 h and 0.5 U/mL rh-INS.Consequently, the healing rate was estimated by capturing images of the wounds at key time points (24,48, and 72 h) using an inverted microscope.

Creation of the K1-TSHR and TPC-1-TSHR Cell Lines Using the Lenti-X Tet-On Advanced System
To multiply the TSH signal reception, cell clones were created to overexpress the TSHR receptor in a controllable manner.Instead of using mutant types of the TSHR gene (many of which have been reported), the wild-type allele was selected since most TSHR mutations are associated with follicular thyroid carcinoma (FTC), in which they occur much more often compared to PTC, wherein mutations are relatively rare [41].Additionally, wild-type TSHR (wt-TSHR) has been reported to possess a high level of constitutive activity in the wild-type form [42]; thus, overexpressing would significantly multiply signal reception and transduction.

Transduction of K1 and TPC-1 Cells Using Lentiviral Vectors
Human wt-TSHR cDNA was subcloned from a pcDNA 3.1 hygro (-) plasmid (a kind gift from Dr. Holger Jaeschke from University Hospital Essen, Germany) to a pLVX-Tight-Puro plasmid [43].This plasmid was used to transfect HEK293T cells (ECACC, Salisbury, UK) to produce lentivirus.Plasmids containing the Lenti-X Tet-on (pLVX-Tight-Puro) advanced system and the embedded cDNA TSHR sequence were constructed using the restriction enzymes BamHI and EcoRI.Subsequently, the transformed plasmids were analyzed using agarose electrophoresis, and the constructed plasmid was isolated and transferred to Escherichia coli DH5a bacteria.The E. coli bacteria were cultured in an LB growth medium containing 100 µg/mL ampicillin; thus, only the transformed clones survived.Plasmids were then isolated using the Monarch ® Plasmid Miniprep Kit (New England Biolabs, Ipswich, MA, USA) and used to transfect HEK293T cells.The culture supernatant containing the viruses produced in the HEK293T cells was used to transfect K1 and TPC-1 cells.Following 24h of incubation with the lentiviruses, the medium was replaced with puromycin-and G418-containing medium at concentrations of 150 µg/mL.After 48 h, only the transformed cells had survived and were still attached.

Transformation Validation Using a Polymerase Chain Reaction
DNA was extracted from lentivirus-transduced cells (K1 and TPC-1), using the Monarch ® Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA, USA), and the quality was determined using the NanoDrop Q500 spectrophotometer (Quawell, CA, USA).PCR was performed to detect the TSHR inserts using the following primers: The housekeeping gene ACTB (β-actin) was used as the reference mRNA.All primers were purchased from IDT (Leuven, Belgium).The cycling conditions consisted of 3 min of denaturation at 95 • C, followed by 30 cycles of 95 • C for 3 s, 60 • C for 30 s and 72 • C for 30 s.The DNA polymerase FIREPol ® and the master mix were from Solis BioDyne (Tartu, Estonia).To assess the induction of TSHR mRNA expression in both transformed cell lines (which were named K1-TSHR and TPC-1-TSHR, respectively) a dose of 3 µg/mL of doxycycline (DOX) was administered, and the procedure described in Section 2.2 was followed.The DOX dose was selected following optimization of the system, as described in previous publications [44].Non-transformed cells (K1, TPC-1) were also monitored as a control group.

Receptor Synthesis Validation with Western Immunoblotting
To assess the TSHR protein levels in both transformed (K1-TSHR and TPC-1-TSHR) and non-transformed (K1 and TPC-1) cells, crude lysates were prepared using Laemmli sample buffer and were subsequently loaded onto 12% polyacrylamide gels for SDS-PAGE analysis.The proteins were then transferred onto an 0.20 µm PVDF Porablot ® membrane (MN, Düren, Germany), and Western blot (WB) analysis was performed using an anti-TSH receptor/TSHR-R rabbit polyclonal IgG antibody (ab58917) (Abcam, Cambridge, UK).Detection was performed using a secondary anti-rabbit IgG HRP-linked goat antibody (CST #7074) and the chemiluminescence system SignalFire™ ECL (CST #6883), both from Cell Signaling Technology (Danvers, MA, USA).As a loading control, the cytoskeletal protein α-tubulin was used, which was detected using an anti-tubulin mouse monoclonal (6-11B-1) IgG2b antibody (#T6793) (Sigma-Aldrich, Darmstadt, Germany), and a secondary anti-mouse IgG HRP-linked horse antibody (CST #7076) from Cell Signaling Technology (Danvers, MA, USA).As a housekeeping gene, α-tubulin has been found to remain stably expressed during all treatments, and its accumulation was relatively similar among the different cell clones studied.The normalization was performed using a plugin for ImageJ (version.2.9.0) created by Suarez-Arnedo (2020) [45].

Proliferation and Migration Assays of K1-TSHR and TPC-1-TSHR Cells
To determine alterations in the proliferation rate descending from TSHR overexpression, the cells were stimulated with doxycycline for 24 h to synthesize an excessive amount of the receptor, as had been demonstrated using qRT-PCR and WB.During the initial 24-h settling period of the proliferation assays, doxycycline was present (at a concentration of 3 µg/mL) to achieve TSHR overexpression.Following the incubation procedure with the hormones (as described in Section 2.3), the cell number was estimated using the crystal violet assay.To assess possible interactions between the doxycycline and the hormones, two sets of control experiments were performed: (a) non-transformed cells (K1, TPC-1) were exposed to doxycycline in the same way as transformed cells (K1-TSHR, TPC-1-TSHR); (b) transformed cells were also assessed in the absence of a doxycycline pre-treatment, theoretically functioning as naïve K1/TPC-1 cells.

Statistical Analysis
To compare the number of cells among the different concentrations of rh-TSH or rh-INS, a one-way ANOVA test was used, followed by Sidak's multiple comparisons test.The results are displayed as means ± the standard error of the mean (SEM).Prism 8 (Graphpad, La Jolla, CA, USA) for Windows was used for the statistics test and for the generation of graphs.Statistical significance was set at a p-value of <0.05.

Effects of Escalating Rh-TSH Concentrations on Thyroglobulin Expression and Proliferation and the Migration Rates of K1 and TPC-1 Cells
Both cancerous cell lines under the culture conditions applied expressed low levels of Tg mRNA, which were quantified using qRT-PCR.The transcription of the Tg gene was used as a marker of thyroidal phenotype maintenance, and any differences in Tg expression were theorized to be the result of rh-TSH and insulin administration.Since both cell lines synthesized low levels of Tg, the detection of the thyroglobulin protein using Western blots or ELISA kits was not feasible; thus, we settled on qRT-PCR results.The low expression was verified by the high C t numbers observed in all qRT-PCR experiments.K1 cells were found to synthesize slightly lower levels of Tg compared to TPC-1 cells; nonetheless, both cell lines' expression levels were low compared to bibliographically recorded information about tumors from PTC patients, where Tg levels were comparable to those of normal thyroid tissue [46].To study the cells' response to rh-TSH stimulation, we incubated them with a wide spectrum of hormone doses (10-100 µIU/mL) to assess their activity regarding Tg synthesis.Stimulation with rh-TSH led to a dose-dependent increase in the Tg mRNA expression of both K1 and TPC-1 cells, indicating that rh-TSH indeed activates the thyroid-specific signaling pathways that lead to Tg expression.Two (clinically relevant) representative doses are presented here (10 and 100 µIU/mL) and the respective statistical significance is indicated (Figure 1a) (p-value < 0.01).Regarding proliferation, K1 cells were incubated for 48 h with increasing concentrations of rh-TSH of 0, 1, 5, 10, 20, 50, and 100 µIU/mL in high-glucose DMEM (Figure 2a).A concentration of 2.5% FBS was used in all the proliferation experiments, as it was determined to be the minimum dose needed for sustaining cell survival and proliferation, thereby avoiding the possible masking effects of rh-TSH action by serum-contained bovine TSH (b-TSH) or other hormones (Figure A1a,b).Iodine intake has been found to be a limiting factor in thyroid cell proliferation (K1 and TPC-1) in in vitro settings [37]; therefore, it could not be absent.According to our data, no discernible differences in cell num- Regarding proliferation, K1 cells were incubated for 48 h with increasing concentrations of rh-TSH of 0, 1, 5, 10, 20, 50, and 100 µIU/mL in high-glucose DMEM (Figure 2a).A concentration of 2.5% FBS was used in all the proliferation experiments, as it was determined to be the minimum dose needed for sustaining cell survival and proliferation, thereby avoiding the possible masking effects of rh-TSH action by serum-contained bovine TSH (b-TSH) or other hormones (Figure A1a,b).Iodine intake has been found to be a limiting factor in thyroid cell proliferation (K1 and TPC-1) in in vitro settings [37]; therefore, it could not be absent.According to our data, no discernible differences in cell number were documented between the various rh-TSH concentrations; therefore, a range of higher concentrations between 100 and 1000 µIU/mL was tested (Figure 2b).The action of rhTSH was examined under various conditions, such as in the presence or absence of insulin, as shown in all graphs, prolonged exposure to the hormones (72 h) (Figure 1c), and with the replenishment of the cell medium every 24 h (to supply the cells with an abundance of rh-TSH) (Figure 2d).Even under those conditions, rh-TSH was incapable of significantly inducing proliferation compared to the control group, which consisted of cells cultured with 10% FBS.This group had elevated proliferation rates compared to the rest of the samples and this activity was a direct consequence of increased serum levels, which contain adequate quantities of pro-proliferative molecules to sustain continuous mitoses, mainly IGFs and iodine salts.The same screening conditions were used to monitor the cancerous cell line TPC-1 (Figure 2e-h) and the results showed that rh-TSH failed to induce cell proliferation at any tested concentration (0-1000 µIU/mL), regardless of the presence of insulin.All insulin-treated samples showed significantly elevated cell numbers compared to the insulin-untreated group, whereas the rh-TSH concentration did not show an additive effect on cell numbers (Figure 2).To examine the effects of rh-TSH/insulin on cancer cell migration, a wound-healing assay was used.The results showed that rh-TSH was incapable of significantly inducing migratory effects on any of the two PTC cell lines, whether insulin was present or not (Figure 3a,b).The assays were performed within a 72-hour timeframe, and FBS was present in all samples since, in its absence, the cells do not migrate or multiply to cover To examine the effects of rh-TSH/insulin on cancer cell migration, a wound-healing assay was used.The results showed that rh-TSH was incapable of significantly inducing migratory effects on any of the two PTC cell lines, whether insulin was present or not (Figure 3a,b).The assays were performed within a 72-h timeframe, and FBS was present in all samples since, in its absence, the cells do not migrate or multiply to cover wounds.However, the concentration of the serum was maintained at a minimum (2.5%) to reduce the masking effects on rh-TSH.Lower FBS concentrations (1%) have been used by other groups in cell cultures of K1 and TPC-1 cells [40]; however, in these experiments, the assay lasted 48 h, and no report on the expression of thyroid markers exists.A complete matrix of the images from all concentrations monitored and all time points can be found in Appendix C (Figure A3a,b).

Creation of TSHR-Overexpressing PTC Cells and TSHR Synthesis Determination
In baseline conditions, qRT-PCR showed that both K1 and TPC-1 cells express low levels of the TSHR mRNA (Figure 4a).This low expression has also been verified by other groups [23,27,28,47], while some others even cite TSHR expression as being undetectable [48][49][50].Low TSHR expression allows only for limited stimulation by circulating TSH molecules and this was theorized to be a great limitation of the hormone's role in the study.Therefore, to eliminate the problem of low TSHR levels, we generated clones of K1 and TPC-1 cells with controllable wt-TSHR expression, using the Lenti-X Tet-on Advanced system.The wild-type receptor was selected due to its prevalence in PTC patients and its increased constitutive activity, compared to many mutations [41,42].Plasmids were constructed containing the wild-type TSHR gene, and successful construction was verified with DNA agarose gel electrophoresis (Figure A2a).Successful transfection and subsequent transformation were verified using PCR to detect copies of the TSHR cDNA insert.Both cell lines had been successfully transformed; thus, the insert was successfully embedded inside the genome (Figure A2b).The transformed cells were named K1-TSHR and TPC-1-TSHR, respectively.Following successful embedment, the expression of TSHR should be inducible through the addition of doxycycline.In order to confirm this finding, 3 µg/mL of doxycycline (DOX) was introduced for 24 h, and the mRNA levels were assessed by qRT-PCR.The K1-TSHR cells reached an almost 25-fold level of overexpression, and the TPC-1-TSHR cell line increased its mRNA TSHR levels by 60 times more than the naïve cell line (Figure 4a).To further confirm that the observed changes at the mRNA level Cells were seeded into six-well microplates, and after the formation of a confluent monolayer, scratches of equal dimensions were made using a pipette tip.Subsequently, the old medium and the debris were aspirated and replaced with a new one containing 2.5% FBS and the designated doses of the hormones.Photographs were taken every 24 h using a camera mounted on a microscope (magnification ×100).(a) Changes were not observed in either K1 or (b) TPC-1 cells following treatment with a high dose of 100 µIU/mL, and the same phenomenon was documented on (c) K1-TSHR and (d) TPC-1-TSHR cells.

Creation of TSHR-Overexpressing PTC Cells and TSHR Synthesis Determination
In baseline conditions, qRT-PCR showed that both K1 and TPC-1 cells express low levels of the TSHR mRNA (Figure 4a).This low expression has also been verified by other groups [23,27,28,47], while some others even cite TSHR expression as being undetectable [48][49][50].Low TSHR expression allows only for limited stimulation by circulating TSH molecules and this was theorized to be a great limitation of the hormone's role in the study.Therefore, to eliminate the problem of low TSHR levels, we generated clones of K1 and TPC-1 cells with controllable wt-TSHR expression, using the Lenti-X Tet-on Advanced system.The wild-type receptor was selected due to its prevalence in PTC patients and its increased constitutive activity, compared to many mutations [41,42].Plasmids were constructed containing the wild-type TSHR gene, and successful construction was verified with DNA agarose gel electrophoresis (Figure A2a).Successful transfection and subsequent transformation were verified using PCR to detect copies of the TSHR cDNA insert.Both cell lines had been successfully transformed; thus, the insert was successfully embedded inside the genome (Figure A2b).The transformed cells were named K1-TSHR and TPC-1-TSHR, respectively.Following successful embedment, the expression of TSHR should be inducible through the addition of doxycycline.In order to confirm this finding, 3 µg/mL of doxycycline (DOX) was introduced for 24 h, and the mRNA levels were assessed by qRT-PCR.The K1-TSHR cells reached an almost 25-fold level of overexpression, and the TPC-1-TSHR cell line increased its mRNA TSHR levels by 60 times more than the naïve cell line (Figure 4a).To further confirm that the observed changes at the mRNA level of TSHR were indeed translated into differential protein expression, TSHR protein accumulation (after stimulation of both cell lines with DOX) was analyzed by Western immunoblotting.The cancerous K1-TSHR overexpressed the receptor by four times and the TPC-1-TSHR cancerous cells by eight times, respectively (Figure 4b,c).After selection of the transformed clones using the appropriate antibiotics, incubation with a dose of 3 µg/mL DOX was introduced to the transformed clones to activate the Tet-On Advanced system.Following 24 h of incubation, total RNA was isolated, cDNA was constructed, and qRT-PCR was performed.Relative expression was assessed using Livak's model.The K1 cell line is annotated with red bars, while the K1-TSHR cells are represented with brown bars.The TPC-1 cell line appears as a green bar, while the TPC-1-TSHR cells appear as light blue bars.Each bar represents the average of three experimental values (n = 3), and the error bars refer to the standard error (SEM).** Corresponds to a p-value of <0.01; ns corresponds to "non-statistically significant".Beta-actin was used as a reference gene to normalize the data.(c) Following 24 h of incubation with 3 µg/mL DOX, the cells were lysed, and the crude protein extracts were analyzed using SDS-PAGE on 12% polyacrylamide gels.Western blot (WB) analysis was performed to detect the TSHR protein band.Using ImageJ, the WB results were quantified (n = 3).*** corresponds to a p-value of <0.001; ns corresponds to "non-statistically significant".Representative Western blot images are shown of (d) K1 (e) and TPC-1.The cytoskeletal protein α-tubulin was used to normalize the results.Due to the increased TSHR synthesis, a lower amount of K1-TSHR/TPC-1-TSHR total protein extract was loaded in the gels to avoid overexposure.The graphs were prepared after normalization with α-tubulin and with consideration of the initial difference in protein quantity.Original Western Blots can be found at Supplementary Materials.

Effects of Rh-TSH Concentrations, on Thyroglobulin Expression, Proliferation, and the Migration of K1-TSHR and TPC-1-TSHR Cells
Transformed K1-TSHR and TPC-1-TSHR cells were assessed for the maintenance of the thyroidal phenotype, using Tg expression as a marker.Additionally, various rh-TSH concentrations (10-100 µIU/mL) were administered (both to DOX-treated and untreated transformed cells) to assess whether the overexpression of TSHR would affect Tg synthesis.To induce TSHR overexpression, a pre-treatment with 3 µg/mL DOX for 24 h was used; subsequently, the cells were incubated for 48 h with the designated hormone dose (rh-TSH).Finally, to determine Tg expression levels, qRT-PCR was used.K1-TSHR cells exhibited an almost 4-fold increase (p < 0.01) in Tg expression (at baseline levels), while incubation with 100 µIU/mL rh-TSH led to a 9-fold increase (p < 0.001) (Figure 5a).Regarding TPC-1-TSHR cells, at baseline levels, a five-fold increase in TG synthesis was documented, and further dose escalation showed results comparable to those of K1 cells (Figure 5b).
Proliferation and migration experiments were carried out on transformed K1-TSHR and TPC-1-TSHR cells after pre-treatment with 3 µg/mL DOX to overexpress the receptor.Comparing the DOX-treated cells to the untreated group, which served as the control group, overall, slightly lower cell numbers in the DOX-treated cells were observed (Figure 6b,e), which were justified as a mild cytostatic consequence of DOX during the overexpression induction period [51].The same cytostatic effect was observed on the non-transformed cells (Figure 6c,f).Although the dose used during the transduction experiments is far lower than for cytotoxic levels, the presence of DOX was expected to mildly affect the proliferation rate, which could occur due to low FBS concentrations.To guarantee that the effects of rh-TSH were not being masked by DOX, the K1-TSHR and TPC-1-TSHR cells were also pre-treated with 3 µg/mL DOX, and their proliferation was assessed.As shown in the figure, similar results were obtained (Figure 6a,d).The positive control (10% FBS) used in the study, regardless of the slightly lower proliferation rate resulting from DOX, exhibited successful proliferation that was far greater than that of FBS-starved cells.Therefore, the presence of DOX was not theorized as being responsible for rh-TSH ineffectiveness or masking in promoting mitosis since, in the case of elevated FBS, it did not act as the limiting factor.Insulin treatment (rh-INS) caused all samples to demonstrate increased proliferation rates; however, this was observed to be independent of the rh-TSH doses.Even when TSHR was being overexpressed, rh-TSH did not indicate a dose-dependent mode of action when administered as the sole pro-proliferation factor.FBS was found to be the most important parameter, and its effects were further amplified following treatment with insulin.sis.To induce TSHR overexpression, a pre-treatment with 3 µg/mL DOX for 24 h was used; subsequently, the cells were incubated for 48 h with the designated hormone dose (rh-TSH).Finally, to determine Tg expression levels, qRT-PCR was used.K1-TSHR cells exhibited an almost 4-fold increase (p < 0.01) in Tg expression (at baseline levels), while incubation with 100 µIU/mL rh-TSH led to a 9-fold increase (p < 0.001) (Figure 5a).Regarding TPC-1-TSHR cells, at baseline levels, a five-fold increase in TG synthesis was documented, and further dose escalation showed results comparable to those of K1 cells (Figure 5b).All the samples were analyzed using Livak's model, and beta-actin was used as a reference gene to Regarding migration potential and wound-healing capacity, the scratch tests revealed that stimulation with rh-TSH (with or without rh-INS) did not cause any changes (Figure 3c,d), even when TSHR was overexpressed.A complete matrix of the images from all concentrations monitored and all time points can be found in Appendix C (Figure A4).

Discussion
Herein, we examine for the first time the effects of clinically relevant concentrations of recombinant human TSH (Thyrogen ® ) on the proliferation and migration of papillary cancer thyroid cell lines, in contrast with previous studies that mostly employed bovine TSH.Surprisingly, we found that the administration of escalating doses of rh-TSH, like those found in plasma, did not increase the cell number or migration rate in K1 and TPC-1 papillary thyroid carcinoma cell lines, which is a fairly new notion since only a few groups have come up with similar conclusions.Since TSH signaling significantly depends on the level of thyroid cell (de)differentiation, we assessed the expression of thyroid markers following administration of rh-TSH, and we found that it tended to increase the levels Regarding migration potential and wound-healing capacity, the scratch tests revealed that stimulation with rh-TSH (with or without rh-INS) did not cause any changes (Figure 3c,d), even when TSHR was overexpressed.A complete matrix of the images from all concentrations monitored and all time points can be found in Appendix C (Figure A4).

Discussion
Herein, we examine for the first time the effects of clinically relevant concentrations of recombinant human TSH (Thyrogen ® ) on the proliferation and migration of papillary cancer thyroid cell lines, in contrast with previous studies that mostly employed bovine TSH.Surprisingly, we found that the administration of escalating doses of rh-TSH, like those found in plasma, did not increase the cell number or migration rate in K1 and TPC-1 papillary thyroid carcinoma cell lines, which is a fairly new notion since only a few groups have come up with similar conclusions.Since TSH signaling significantly depends on the level of thyroid cell (de)differentiation, we assessed the expression of thyroid markers following administration of rh-TSH, and we found that it tended to increase the levels of Tg mRNA, a direct proof of successful stimulation.Due to the low expression of TSHR in these cell lines (Figure 4a), we successfully transformed them to overexpress TSHR (Figure 4b) and assessed the mutant clones.Once again, the same escalating doses of rh-TSH were unable to induce proliferation or migration of the cells, regardless of the theoretically increased signal transduction through the TSHR.Moreover, all experiments were conducted under various such as the presence or absence of insulin (which is theorized to be one of the most important synergistic hormones), the administration of a wide range of doses, and the study of different time frames, as well as culture medium replenishments every 24 h.All these conditions did not produce substantial evidence regarding the activating role of TSH on the studied cell functions, possibly indicating an inferior role for TSH compared to other parameters.
Although there is a prevailing perception that TSH induces the growth and proliferation of follicular thyroid cells, there is limited evidence as to whether the proliferation is a direct consequence of TSH stimulation, or whether other ligands that act synergistically with TSH activate the TSHR-dependent signaling cascade and lead to observable increased mitotic activity.In vitro studies have primarily been conducted using cultures derived from animal cell lines, with only a few studies focusing on human cell cultures that include both normal and malignant thyrocytes.These studies have investigated the effects of various doses of bovine/cattle TSH, mainly on cellular responses, particularly regarding cell proliferation.However, the results obtained from these studies have been inconsistent, suggesting that the responses of thyroid cells to TSH treatment can vary widely across different experimental conditions and cell types [19][20][21]23,26,[52][53][54].In one study, researchers investigated the effects of escalating doses of b-TSH (1, 10, and 100 mU/mL) on growth and invasion in primary cell cultures of follicular and papillary thyroid carcinomas [52].Specifically, they analyzed three follicular thyroid carcinoma (FTC) cell lines that were derived from one patient, comprising one from a primary tumor and two from metachronous metastases that originated from a lymph node and lung metastasis.Additionally, they examined two PTC cell lines from two patients who had not experienced disease recurrence.Notably, the maximal effect on proliferation and invasion was observed at a dose of 10 mU/mL (equivalent to 1000× of that found in human plasma), while a dose of 100 mU/mL was found to inhibit proliferation.Intriguingly, the response of the five cell lines varied in terms of growth and invasion at the same b-TSH dose, with PTC lines exhibiting the maximal response, followed by FTC lines, and even smaller responses observed in metastatic lines, while inhibition remained consistent [52].In another in vitro study, the authors found that the growth of papillary thyroid cancer cells and adenomatous goiter cells was differentially regulated.Both cell lines were found to express functional TSH receptors.When treated with b-TSH at a dose of 10 µU/mL (a dosage similar to that used in our study) and with or without IGF1, the proliferation of cancerous thyrocytes was inhibited, while that of human adenomatous goiter cells was stimulated.Intriguingly, the activation of TK receptors by various growth factors, such as EGF, b-FGF, and IGF1, along with stimulation by E2 (at subclinical concentrations), promoted proliferation in both cancerous and goitrous cells [21].More recently, a single application of 100, 1000, and 10 4 µIU/mL of cattle TSH in human PTC cells (TPC-1) increased the proliferation rate in a dose-dependent manner by promoting the transition of TPC-1 cells from the G 1 phase to the S phase and by markedly increasing the mRNA and protein expression of cyclin D1 [23].The contradictory results observed in these various in vitro studies could be attributed to several factors, such as the TSH dose used, discrepancies in the specific culture systems used, including variations in cell culture media, supplements, and culture conditions, incubation time, context (normal or cancer cell line), and the method of growth assessment.As FBS contains endogenous TSH (b-TSH), insulin, dissolved iodine salts, and related cofactors that are required for thyroid cell survival, and since the FBS batches vary significantly, we tried to minimize the effects of these factors by using the minimum required concentration of FBS in our experiments.
It is also noteworthy that most of the published studies have not adequately evaluated the expression of TSHR in the various cell line models.Differences in the media used and multiple passages of cell lines can sometimes have deleterious or silencing effects on the thyroid-like phenotype and the expression of TSHR, which on some phenotypic characterizations have even been reported as being absent [49,50].Many studies even report that important thyroid markers like TSHR, Tg, thyroid peroxidase (TPO), and sodiumiodide-symporter (NIS) are not even expressed by the cells, thus creating the question of whether these cells can still be considered a valid thyroid cell model [48].It is known that the expression of TSHR in primary cultures is 100-fold lower than in intact thyroid cells in vivo [55].To overcome this obstacle, at least partially, we transformed the two cell lines to overexpress TSHR as a way to secure stable and increased stimulation by TSH.The overexpression was controlled by the presence of doxycycline, and the transformed cells (K1-TSHR and TPC-1-TSHR) were assessed for the same functions as previously noted.Interestingly, no significant changes were observed following stimulation with rh-TSH and insulin, exactly like the non-transformed clones.The migration rates were also unchanged, and the pro-proliferative effects of insulin (when administered alone) were also documented.Our findings are consistent with a prior study wherein 10 mU/mL of b-TSH failed to induce proliferation in human thyroid follicular carcinoma cells that were transfected to overexpress functional TSHR [56].
Besides the expression of TSHR per se, it is also important to note its functionality and how relevant mutations of the receptor and other important molecules in the signaling pathway may affect signal transduction and, ultimately, cell proliferation.The cell lines that were used in the present work are not known to carry any TSHR mutations, and the TSHR form that is expressed is the wild-type form.The K1 and TPC-1 cell lines are derived from papillary thyroid carcinoma and carry, respectively, the following genetic aberrations: BRAF V600E and RET/PTC1 fusion [29,57].TPC-1 cells carry the RET/PTC1 fusion, which can lead to elevated Met proto-oncogene expression, resulting in an invasive phenotype [58].Regarding BRAF alleles, TPC-1 cells are homozygous to the wild-type gene while K1 cells carry one allele of the BRAF V600E mutation, which can lead to increased signal transduction through the Ras-Raf-MAPK pathway [29,59].Such mutations have been reported to be prevalent among PTC patients and can significantly affect the progression of the disease since they are correlated with more aggressive phenotypes [60].Both cell lines have been reported to be genetically identical regarding thyroidal characteristics [61].Both at baseline and at overexpressed levels of wild-type TSHR, we saw practically no effect on the proliferation of cells descending from rh-TSH stimulation, regardless of the conditions used herein.Additionally, the overexpression of TSHR has been recently tested in TPC-1 cells; however, this is not in an inducible manner as herein presented [47].In a preprint by Lin et al. in 2022, it is reported that TPC-1 expressed very low levels of the TSHR mRNA, and the cell line is transfected with a TSHR-carrying pCDH-CMV-MCS-EF1-Puro (CD510B) plasmid to overexpress the receptor [47].The authors reported that TSHR overexpression suppressed proliferation; nonetheless, they do not mention any stimulation with TSH (of a bovine or human recombinant nature).It is hypothesized that TSHR elevated the phosphorylation levels of IkB, which, in turn, reduced NF-κB signaling, thus reducing proliferation [47].However, to this day, no further investigation of the role of overexpressed TSHR has been performed.Moreover, it would be plausible to investigate further the effect of various TSH concentrations, not only in wtTSHR but also in various mutated forms of TSHR that may affect intracellular signaling and, thus, the proliferation rate, as well as the study of more co-factors.
Further assessment of the signaling pathways downstream of the TSHR, as well as the synergistic actions of other receptors, is also warranted under these conditions and can be the subject of future studies.In order to perform a preliminary investigation of rh-TSH interaction with other circulating hormones, we administered insulin, one of the most important hormones that act synergistically with TSH [26,[62][63][64].Besides insulin, insulin-like growth factors (IGFs) are part of a protein family with many roles in both physiological functions and disease [65,66].Both IGFs and insulin are known to bind the insulin receptor (IR), as well as the insulin-like growth factor receptors I and II (IGF1R, IGF2R), which can dimerize with the TSHR and transduce signals.Insulin has a lower affinity for IGF1R compared to IGF1; however, higher concentrations of insulin have been found to successfully activate the receptor [33][34][35].Specifically, insulin-like growth factor I receptor (IGF1R) is a transmembrane tyrosine kinase receptor involved in growth and development [67].It seems that there is a crosstalk between the TSH/TSHR and insulin/IGF1/IGF1R pathways in thyroid cell proliferation and differentiation [63,64,[68][69][70][71][72][73].FBS contains both bovine IGF1 and bovine insulin, both of which are useful to maintain the normal growth of the cell lines since they are equipotent to human hormones [74,75]; however, in an attempt to overstimulate IGF1R, which has significant crosstalk with TSHR, we modified the culture medium to contain a high concentration of recombinant human insulin (Humalog ® ).In agreement with our experimental design and data, in most of the in vitro studies, IGF1 or high insulin levels are required for the culture to achieve receptor activation [26,76].According to our experiments, the addition of insulin at a concentration of 0.5 U/mL in the medium on top of rh-TSH increased the cell number, but not in a rh-TSH dose-dependent manner.
TSH, besides proliferation, modulates thyroid hormone synthesis and secretion by regulating the expression of various thyroid-specific such as TSHR, Tg, TPO, and NIS, all of which are considered markers of thyroid cell phenotype, and their maintenance guarantees that no de-differentiation has occurred.Our findings indicated that the treatment of K1 and TPC-1 papillary thyroid cancer cells with increasing concentrations of rh-TSH led to a gradual increase in expression of the Tg gene, with peak mRNA expression levels at the highest dose.This was also evident in the transformed clones, where Tg levels were found to be almost five-fold higher compared to their non-transformed counterparts and retained a dose-response expression pattern.Our results seem to be consistent with previous studies where the treatment of cultured thyroid cells with b-TSH stimulated the TPO and Tg mRNA levels in a doseand time-dependent manner [77,78].Additionally, this verified the successfully increased TSHR-mediated signal transduction taking place in transformed cells.A limitation of our study is that it is based on in vitro experiments and employs cell lines that may gradually lose some of the properties of the original thyroid tissue, which may occur due to mutations and/or epigenetic silencing resulting from selection pressure during prolonged cell culture [49,79].To at least partially address this concern, we evaluated our cell lines for the preservation of the thyroidal phenotype by assessing Tg mRNA expression, observing that the cells retained some features of their original phenotype since the signaling pathways (and enzymes) that regulate Tg synthesis are specific to thyroid cells.However, it is noteworthy that under basal conditions, TSHR mRNA expression was low in our cancerous cell lines, while normal thyroid tissue showed significantly higher expression of the receptor.To overcome this limitation, we engineered both cell lines to overexpress the thyrotropin receptor.To strengthen our findings, further investigations are warranted using additional cell types that overexpress wild-type as well as mutant TSHR variants (various versions of constitutively active or inactive variants, as aforementioned) [43] or utilizing more physiologically relevant cell systems, such as primary cultures of thyroid gland organoids [80] from cancerous thyroid tissue.Additionally, in future studies, it is imperative to assess the functionality of the overexpressed TSHR by assessing the activation status of downstream signaling pathways that were not assessed in this study, given that no effect was seen in proliferation, thereby possibly expanding our understanding of the synergistic roles of IGFs and their receptors.

Conclusions
In conclusion, we have shown that rh-TSH in clinically relevant doses cannot induce proliferation and migration in papillary cancerous cell lines under various conditions.Further research is warranted to dissect the molecular mechanisms underlying these effects.There is an ongoing debate in the literature about whether TSH suppression during the follow-up of PTC patients prevents the progression or recurrence of the disease, especially in intermediate and high-risk patients [6,10].Our data support the strategy of avoiding TSH suppression and, thus, iatrogenic thyrotoxicosis.Consequently, our results could translate into better management of the treatment of PTC patients in the future.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers16142604/s1,Gel and Blot Images.Cells were seeded into six-well microplates and, after the formation of a confluent monolayer, scratches of equal dimensions were made using a pipette tip.Subsequently, the old medium and the debris were aspirated and replaced with new medium containing 2.5% FBS, a range of rh-TSH doses from 5 µIU/mL to 100 µIU/mL, and 0.5 U/mL rh-INS.Photographs were taken every 24 h using a camera mounted on a microscope (100× magnification).No changes were observed in either the non-transformed cells (a) or transformed cells (b).

Figure 1 .
Figure 1.Thyroglobulin (Tg) gene expression determination of K1 and TPC-1 cells.(a) Using qRT-PCR, the baseline expression levels of the Tg mRNA of K1 (red bar) and TPC-1 cells (green bar) were assessed.(b) Following 48 h of treatment with the designated doses of rh-TSH, qRT-PCR was used to assess the effects of the hormone on Tg mRNA expression in K1 cells, while the same assay was performed in TPC-1 cells as well (c).All the samples were analyzed using Livak's model, and betaactin was used as a reference gene to normalize the data.All experiments were conducted in triplicate (n = 3), and each bar represents the average of the three experimental values.The error bars refer to the standard error of the mean (SEM).** Corresponds to a p-value of < 0.01; *** corresponds to a p-value of < 0.001.

Figure 1 .
Figure 1.Thyroglobulin (Tg) gene expression determination of K1 and TPC-1 cells.(a) Using qRT-PCR, the baseline expression levels of the Tg mRNA of K1 (red bar) and TPC-1 cells (green bar) were assessed.(b) Following 48 h of treatment with the designated doses of rh-TSH, qRT-PCR was used to assess the effects of the hormone on Tg mRNA expression in K1 cells, while the same assay was performed in TPC-1 cells as well (c).All the samples were analyzed using Livak's model, and beta-actin was used as a reference gene to normalize the data.All experiments were conducted in triplicate (n = 3), and each bar represents the average of the three experimental values.The error bars refer to the standard error of the mean (SEM).** Corresponds to a p-value of <0.01; *** corresponds to a p-value of <0.001.

Figure 2 .
Figure 2. Proliferation assay of K1 and TPC-1 cells treated with rh-TSH and/or rh-INS.Cells were seeded into microplates, and after 24 h of settling and attachment, the old medium was aspirated and replaced with a new one containing 2.5% FBS, and the designated doses of the hormones (rh-TSH and rh-INS) were added for 48 h (or 72 h).Wherever insulin is annotated (rh-INS), a concentration of 0.5 U/mL was used.As a positive control (striped bars, annotated as C) for elevated cell proliferation rates, a 10% FBS-containing medium was used that has endogenous b-TSH, b-IGF1, insulin, and other cofactors.The vehicle-treated cells (annotated as Vehicle) were cultured in a 2.5% FBS-containing medium without added rh-TSH or rh-INS.The red bars represent rh-INS-untreated K1 cells, while the orange bars represent rh-INS-treated K1 cells.(a) A range of rh-TSH concentrations from 5 µIU/mL to 100 µIU/mL was initially tested in K1 cells, (b) as well as a broader spectrum from 50 µIU/mL to 1000 µIU/mL.(c) Assays were performed over a 72-h timeframe.(d) To test whether TSHR was rapidly absorbed/decomposed or inactivated by the cells, we replenished the hormone-containing medium after 24 h and let the cells grow.(e-h) The aforementioned conditions were replicated for the TPC-1 cell line.The dark green bars represent rh-INS-untreated TPC-1 cells, while the light green bars represent rh-INS-treated TPC-1 cells.All experiments (a-h) were conducted in triplicate (n = 3), and the error bars represent the standard error of the mean (SEM).Data analysis was performed using multiple t-tests, and no significant differences between the different rh-TSH concentrations were detected.Insulin-treated cells exhibited an overall increased cell number, which was statistically significant, as shown by a paired t-test (p-value < 0.001).*** Corresponds to a p-value of <0.001.

Figure 3 .
Figure 3. Wound-healing assay of K1, TPC-1, K1-TSHR, and TPC-1-TSHR cells treated with rh-TSH.Cells were seeded into six-well microplates, and after the formation of a confluent monolayer, scratches of equal dimensions were made using a pipette tip.Subsequently, the old medium and the debris were aspirated and replaced with a new one containing 2.5% FBS and the designated doses of the hormones.Photographs were taken every 24 h using a camera mounted on a microscope (magnification ×100).(a) Changes were not observed in either K1 or (b) TPC-1 cells following treatment with a high dose of 100 µIU/mL, and the same phenomenon was documented on (c) K1-TSHR and (d) TPC-1-TSHR cells.

Figure 3 .
Figure 3. Wound-healing assay of K1, TPC-1, K1-TSHR, and TPC-1-TSHR cells treated with rh-TSH.Cells were seeded into six-well microplates, and after the formation of a confluent monolayer, scratches of equal dimensions were made using a pipette tip.Subsequently, the old medium and the debris were aspirated and replaced with a new one containing 2.5% FBS and the designated doses of the hormones.Photographs were taken every 24 h using a camera mounted on a microscope (magnification ×100).(a) Changes were not observed in either K1 or (b) TPC-1 cells following treatment with a high dose of 100 µIU/mL, and the same phenomenon was documented on (c) K1-TSHR and (d) TPC-1-TSHR cells.

Figure 4 .
Figure 4. TSHR mRNA and protein expression levels following transformation with the Lenti-X Tet-on Advanced system.(a) Using qRT-PCR, the baseline expression levels of the TSHR mRNA of

Figure 5 .
Figure 5. Thyroglobulin (Tg) gene expression determination of K1-TSHR and TPC-1-TSHR Cells.(a)Following 24 h of treatment with 3 µg/mL DOX, K1-TSHR-transformed cells were incubated for 48 h with the designated doses of rh-TSH.qRT-PCR was used to assess the effects of the hormone on Tg mRNA expression in K1 cells, while the same assay was performed on TPC-1 cells as well (b).All the samples were analyzed using Livak's model, and beta-actin was used as a reference gene to

Figure 5 .Figure 6 .Figure 6 .
Figure 5. Thyroglobulin (Tg) gene expression determination of K1-TSHR and TPC-1-TSHR Cells.(a) Following 24 h of treatment with 3 µg/mL DOX, K1-TSHR-transformed cells were incubated for 48 h with the designated doses of rh-TSH.qRT-PCR was used to assess the effects of the hormone on Tg mRNA expression in K1 cells, while the same assay was performed on TPC-1 cells as well (b).All the samples were analyzed using Livak's model, and beta-actin was used as a reference gene to normalize the data.All experiments (a,b) were conducted in triplicate (n = 3) and each bar represents the average of the three experimental values.The error bars refer to the standard error of the mean (SEM).*** Corresponds to a p-value of <0.001.

Figure 6 .
Figure 6.Proliferation assay of K1-TSHR and TPC-1-TSHR cells treated with rh-TSH and/or rh-INS.The transformed cells were seeded into microplates in a medium containing 3 µg/mL doxycycline (DOX), then, after 24 h of settling, attachment, and TSHR overexpression, the old medium was aspirated.An assay medium containing 2.5% FBS and the designated doses of the hormones (rh-TSH, h-INS) were added and left for 48 h.Wherever insulin is annotated (rh-INS), a concentration of 0.5 U/mL was used.As a positive control (striped bars, annotated as C) for elevated cell proliferation rates, a 10% FBS-containing medium was used, which has endogenous b-TSH, b-IGF1, insulin, and other cofactors.The vehicle-treated cells (annotated as Vehicle) were cultured in a 2.5% FBS-containing medium without added rh-TSH or rh-INS.A range of rh-TSH concentrations from 5 µIU/mL to 100 µIU/mL was tested in both transformed K1 (K1-TSHR) and TPC-1 (TPC-1-TSHR) cells.(a) The red bars represent rh-INS-untreated K1-TSHR cells, while the orange bars represent rh-INS-treated K1-TSHR cells.(b) As a control experiment, the effects of rh-TSH were also tested in both DOX-untreated (red bars) and DOX-treated (magenta bars) K1-TSHR cells.(c) The effects of DOX were also assessed in (untransformed) K1 cells: the red bars represent DOX-untreated K1 cells, while the purple bars represent DOX-treated K1 cells.The same set of experiments were performed in TPC-1/TPC-1-TSHR cells.(d) The dark green bars represent rh-INS-untreated TPC-1-TSHR cells, while the light green bars represent rh-INS-treated TPC-1-TSHR cells.(e) DOX-untreated TCP-1-TSHR cells are annotated as green bars, while DOX-treated TPC-1-TSHR cells are represented by blue bars.(f) The green bars correspond to DOX-untreated (untransformed) TPC-1 cells, while the blue bars correspond to DOX-treated TCP-1 cells.All experiments (a-f) were conducted in triplicate, and the error bars represent the standard error of the mean (SEM).The data analysis was performed using multiple t-tests, and no significant differences between the different rh-TSH concentrations were detected.Insulin-treated cells exhibited an overall increased cell number, which was statistically significant, as shown by a paired t-test (p-value < 0.001).*** Corresponds to a p-value of <0.001.

Figure A4 .
Figure A4.Wound healing assay of K1-TSHR and TPC-1-TSHR cells.Cells were seeded into six-well microplates and, after the formation of a confluent monolayer, scratches of equal dimensions were made using a pipette tip.Subsequently, the old medium and the debris were aspirated and replaced with new medium containing 2.5% FBS, a range of rh-TSH doses from 5 µIU/mL to 100 µIU/mL, and 0.5 U/mL rh-INS.Photographs were taken every 24 h using a camera mounted on a microscope (100× magnification).No changes were observed in either the non-transformed cells (a) or transformed cells (b).